The Challenge to Deliver Oxaliplatin (l-OHP) to Solid Tumors: Development of Liposomal l-OHP Formulations

Abstract

Oxaliplatin (l-OHP) is a third-generation platinum (Pt) agent approved for the treatment of patients with advanced colorectal cancer. Despite the fact that l-OHP has shown clinical therapeutic efficacy and better tolerability compared with other Pt agents, the use of l-OHP has been limited to clinical settings because of dose-limiting side effects such as cumulative neurotoxicity and acute dysesthesias, which can be severe. In preclinical and clinical studies, our group and several others have attempted the delivery of l-OHP to solid tumors via encapsulation in PEGylated liposomes. Herein, we review these attempts.

1. Introduction

Cancer has been a major global cause of mortality for the past several decades. Platinum (Pt) is one of the most frequently used anticancer agents in clinical settings.^1,2) Both the United States Food and Drug Administration (FDA) and the Japan Pharmaceuticals and Medical Devices Agency (PMDA) have approved three main Pt drugs: cisplatin, carboplatin, and oxaliplatin (l-OHP)¹⁾ (Fig. 1, Table 1). Furthermore, another candidate for approval as a Pt agent, Satraplatin, is currently undergoing clinical trials (Table 1).

Fig. 1. Chemical Structure of Pt Agents; Cisplatin, Carboplatin, and Oxaliplatin

Table 1. Platinum-based Therapeutic Agents in Clinical Used or Candidates Undergoing Clinical Trials

Interventions	Phases	Usage or Conditions	Route	Status	Approval year	Start Date	Comp. Date	NCT Number	Sponsor/Collaborators	Refs.
Cisplatin	Approved	Bladder cancer	IV	Commercialized	1978 (U.S.) 1984 (JP)					80)
		Lung cancer
		Lymphomas
		Ovarian cancer
		Melanoma
		Testicular cancer
Carboplatin	Approved	Ovarian cancer	IV	Commercialized	1989 (U.S.) 1990 (JP)					81)
		Lung cancer
		Head and neck cancer
Oxaliplatin	Approved	Colorectal cancer	IV	Commercialized	2004 (U.S.) 2004 (JP)					82)
		Pancreas cancer
		Gastric cancer
Nedaplatin	Approved	Head and neck cancer	IV	Commercialized	1995 (JP)					83)
		Small cell lung cancer
		Non-small cell lung cancer
		Esophageal cancer
		Bladder cancer
		Testicular tumor
		Ovarian cancer
		Cervical cancer
Miriplatin	Approved	Lipiodolization in hepatocellular carcinoma	IV	Commercialized	2009 (JP)					84)
Satraplatin	II	Metastatic Breast Cancer	Orally	Completed		Nov, 2005	Feb, 2008	NCT00265655	Agennix
	II	Prostate Cancer	Orally	Completed		Dec, 2010	Jan, 2018	NCT01289067	William K. Oh	85–87)
									Prostate Cancer Foundation
	I	Solid Tumors	Orally	Completed		Dec, 2010	May, 2015	NCT01259479	National Cancer Institute (NCI)	88,89)
		Brain Tumors							National Institutes of Health Clinical Center (CC)
		Brain Metastases

As the first approved Pt agent, cisplatin produces strong cytotoxicity in various cancers: bladder cancer,³⁾ lung cancer,⁴⁾ lymphomas,⁵⁾ ovarian cancer,⁶⁾ melanoma,⁷⁾ and testicular cancer.⁸⁾ Its clinical application has been limited, however, due to the induction of severe side effects such as nephrotoxicity, vomiting, peripheral neuropathy,⁹⁾ and the requirement of prior hydration with transfusion, particularly for elderly patients. Carboplatin or nedaplatin is a second-generation approved Pt agent that is used for the treatment of ovarian cancer,⁶⁾ lung cancer,¹⁰⁾ and head and neck cancer.¹¹⁾ When comparing to cisplatin, carboplatin and nedaplatin show an advantage in reduction of side effects, particularly the elimination of nephrotoxic effects. Depending on the type of cancer, however, carboplatin is less potent than cisplatin,¹²⁾ and nedaplatin is limited for approval in regional conditions. Miriplatin is a third-generation Pt agent that was limitedly approved for the treatment of lipiodolization in hepatocellular carcinoma and succeeded to decrease the toxicities induced by cisplatin. Satraplatin is an oral type of lipophilic platinum prodrug and completed in Phase II studies for the treatment of metastatic breast cancer and prostate cancer.

l-OHP is another third-generation Pt agent with a 1,2-diaminocyclohexane (DACH) carrier ligand, which has been approved for the treatment of several cancers: advanced and metastatic colorectal cancers,¹³⁾ pancreas cancer,¹⁴⁾ and gastric cancer.¹⁵⁾ In clinical settings, l-OHP is commonly used in combination with 5-fluorouracil (5-FU) and leucovorin, as a regimen of FOLFOX, or with S-1, which is an oral anticancer drug consisting of tegafur (a prodrug of 5-FU), 5-chloro-2,4-dihydroxypyrimidine (CDHP), and potassium oxonate with a molar ratio of 1 : 0.4 : 1, as a regimen of SOX. Either FOLFOX or SOX is a standard first- or second-line therapeutic regimen for the treatment of patients with metastatic or advanced colorectal cancer.^16,17) The mode of action for l-OHP is the production of cytotoxic effects by directly cross-linking to the guanine base in DNA double-helix strands, which subsequently prevents DNA synthesis and repair¹⁸⁾ as featured in Fig. 2. l-OHP was designed to lessen the severe adverse events caused by other Pt drugs.¹⁹⁾ However, the desired therapeutic effects of l-OHP are limited by its poor rate of accumulation in tumors, its rate of nonselective accumulation in the healthy organs, its inactivation via conjugation with glutathione, and by the induction of drug resistance.^20,21) In addition, in clinical settings, l-OHP is also reported to induce severe cumulative side effects such as sensory neuropathy, acute dysesthesias, nausea, vomiting, diarrhea, and hematologic dyscrasias.^22–25) Accordingly, in order to overcome such limitations, the application of a drug delivery system (DDS) to improve the pharmacokinetics of l-OHP is required.

Fig. 2. Mechanism of Action for Oxaliplatin

2. Systems for the Delivery of Pt Agents to Solid Tumors

To date, many drug delivery techniques have been developed with the ability to endow a drug of interest with desired physical, chemical, and biological properties.^26,27) Drug delivery techniques for Pt agent formulations are currently undergoing clinical trials (Table 2). NC-4016 is made up of polymeric micellar nanoparticles that incorporate DACH-Pt, which is a partial component of l-OHP.²⁸⁾ In in vivo study, NC-4016 produced superior antitumor effects on a subline of HeLa cells, KB, in a xenograft mouse model and did not induce the severe acute cold hypersensitivity that is frequently expressed in patients treated with free l-OHP in clinical settings.²⁸⁾ AP5346 is another type of polymeric conjugates that incorporates DACH-Pt.²⁹⁾ Compared with free l-OHP, AP5346 has shown higher antitumor activity on both a B16 mouse melanoma tumor-bearing model and a 2008 human ovarian carcinoma xenograft mouse model.³⁰⁾

Table 2. Platinum-Based Formulation Candidates Undergoing Clinical Trials

Interventions (company)	Drugs	Formulation	Phases	Conditions	Status	NCT Number	Refs
NC-6004 (NanoCarrier)	Cisplatin	mPEG-b-PGA micelle	III	Pancreatic neoplasms	Completed	NCT02043288
			I/II	Locally advanced and metastatic pancreatic cancer	Completed	NCT00910741
			I/II	Solid tumors	Completed	NCT02240238	90–94)
			I/II	Carcinoma	Completed	NCT03109158
				Squamous cell of head and neck
			I	Head and neck neoplasms	Terminated	NCT02817113
			II	SCCHN	Recruiting	NCT03771820
BP-C1 (Meabco A/S)	Pt (II)	Benzo-polycarbonic acid polymer	I	Metastatic breast cancer	Completed	NCT04298333	95)
				Stage IV breast cancer
			II	Metastatic breast cancer	Completed	NCT03789019	96)
				Stage IV breast cancer
			II	Metastatic pancreatic cancer	Completed	NCT03627390	96)
				Unresectable pancreatic cancer
			II	Metastatic breast cancer	Completed	NCT03603197	97)
				Stage IV breast cancer
			I	Metastatic breast cancer	Completed	NCT01861509	95)
				Stage IV breast cancer
			II	Metastatic breast cancer	Completed	NCT02783794	96)
				Stage IV breast cancer
NC-4016 (NanoCarrier)	DACH-Pt	mPEG-b-PGA micelle	III	Advanced cancer	Completed	NCT03168035	28,98)
				Lymphoma
AP5346 (Access Pharmaceutical, Inc.)	DACH-Pt	HMPA	II	Advanced ovarian cancer	Unknown	NCT00415298	29,30,99)
Lipoplatin (Centre Hospitalier Universitaire Vaudois)	Cisplatin	Liposome	III	Non-small cell lung cancer, gastric cancer	Terminated	NCT02702700	100–103)
Spi-77 (NYU Langone Health)	Cisplatin	Liposome	II	Ovarian cancer	Completed	NCT00004083	104)
			I/II	Osteosarcoma metastatic	Completed	NCT00102531	105)
					Has results
			II	Malignant mesothelioma	Completed	NCT00004033
			I/II	Advanced or refractory solid tumor	Active, Not recruiting	NCT01861496	106–111)
				Metastatic breast cancer
				Prostate cancer and skin cancer
			III	Pancreatic cancer	Completed	NCT00416507
MBP-426 (Mebiopharm Co., Ltd.)	Oxaliplatin	Liposome	I/II	Gastric Adenocarcinoma	Unknown	NCT00964080	70)
				Gastroesophageal Junction Adenocarcinoma
				Esophageal Adenocarcinoma
			I	Cancer	Completed	NCT00355888

Also, reports have indicated the development of a l-OHP-based delivery system that has improved the therapeutic efficacy of l-OHP in preclinical studies. Wang et al.³¹⁾ synthesized a reduction-responsive polymer, poly(1,2,4,5-cyclohexanetetracarboxylic dianhydride-co-hydroxy-ethyl disulfide)-polyethylene glycol, and prepared l-OHP-encapsulating polymer nanoparticles with a structure that contains disulfide bonds. These polymer nanoparticles release the encapsulated l-OHP after accumulation within tumors, because the tumor microenvironment forms a reductive condition owing to the production of glutathione,³²⁾ which achieves a tumor-selective release of l-OHP.³¹⁾ Schueffl et al.³³⁾ designed an albumin-targeting l-OHP-releasing Pt prodrug, which contains two maleimide moieties within a molecule that has the ability to bind to cysteine 34 of serum albumin after intravenous injection. The resultant albumin nanoparticle loading l-OHP can prolong the half-life period of l-OHP in circulation and increase the accumulation of l-OHP in the tumor, resulting in an increase of the antitumor effects of l-OHP. Zhang et al.³⁴⁾ developed mesoporous silica nanoparticles (MSNs) incorporating l-OHP and photosensitizer HCE6 (OH-MSNs). OH-MSNs have a potency of pH-sensitive drug release because photosensitizer HCE6 could enhance its efficacy in acidic condition, which consequently enhance the release of l-OHP under acidic conditions formed by the tumor microenvironment. When combined with photodynamic treatment of an FRH0201 human hilar cholangiocarcinoma xenograft mouse model, this regimen has shown antitumor effects that are superior to those of free l-OHP.³⁴⁾

Despite the existence of several l-OHP-based delivery systems that have been reported to enhance the therapeutic efficacy of l-OHP, however, few of these have undergone clinical trials, and none have yet been approved. Therefore, we felt challenged to design a new l-OHP-based delivery system using liposomes that could enhance the therapeutic efficacy of l-OHP while reducing the systemic side effects that plague the use of free l-OHP treatment.

3. Liposomes for Delivery of l-OHP to Solid Tumors

3.1. Liposomes

Liposomes are submicron-sized phospholipid bilayer vesicles that consist of several types of phospholipids and cholesterol, and are fascinating as potential DDS carriers that could accommodate a variety of drugs with different physicochemical characteristics. These vesicles are controllable in size, hydrophobic/hydrophilic properties, and surface modification with macromolecules such as antibodies,³⁵⁾ ligands,³⁶⁾ and polyethylene glycol (PEG).^37,38) To date, several types of liposomal anticancer drugs have been marketed. A liposomal formulation encapsulating doxorubicin (Doxil^®), approved by the U.S. FDA in November 1995 and the Japan PMDA in February 2007, is a typical example. Doxil^® is modified with PEG on the surface of liposomes, which provides the liposomes with a prolonged retention feature in blood by forming a hydrated layer on its surface and with features that evade interception by the immune system. This PEGylated liposomal formulation avoids the severe adverse events caused by dosing with free doxorubicin such as cardiotoxicity^39,40) and enhances the accumulation of doxorubicin in solid tumors that form a leaky vascular structure, which is called the Enhanced Permeability and Retention (EPR) effect.⁴¹⁾ Liposomal formulations that have been approved by the U.S. FDA and/or the Japan PMDA include daunorubicin-encapsulating liposomes (DaunoXome^®), cytarabine-encapsulating liposomes (Depocyt^®), vincristine-encapsulating liposomes (Marqibo^®), and irinotecan-encapsulating liposomes (Onivyde^®). In addition, a liposomal formulation encapsulating two anticancer agents, daunorubicin and cytarabine, in a single liposome (Vyxeos^®) was first approved in August 2017.⁴²⁾ Overall, liposomes are promising DDS carriers that decrease undesirable side effects while enhancing the therapeutic effects of encapsulated anticancer agents.

3.2. PEGylated Liposomal l-OHP Formulation

Several groups are now working on the development of l-OHP liposomal formulations. For example, Yang et al.⁴³⁾ have shown that intravenous (i.v.) administration of PEGylated liposomal l-OHP significantly enhances the accumulation of l-OHP in tumor tissues via the EPR effect. This has led to a remarkable reduction in the burden of tumors and compared with free l-OHP has prolonged survival in a xenograft tumor-bearing nude mouse model. Zalba et al.⁴⁴⁾ prepared several types of PEGylated liposomes containing l-OHP using different methods and lipid compositions. They showed that a formulation composed of hydrogenated soybean phosphatidylcholine (HSPC) : cholesterol : mPEG₂₀₀₀-distearoylphosphatidylethanolamine (DSPE) (2 : 1 : 0.2 M ratio) was the most stable in vitro and induced improved efficacy of l-OHP in HT-29 tumor-bearing mice. Lipoxal™ is a PEGylated liposome containing l-OHP and composed of HSPC, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), cholesterol, and mPEG₂₀₀₀-DSPE, and is one of the most advanced l-OHP formulations.⁴⁵⁾ In animal studies, Lipoxal™ has shown a reduction in the adverse reactions of l-OHP, with an efficacy that is similar to the free drug. For clinical trials, Lipoxal™ was given in monotherapy as an i.v. infusion once a week to 27 patients with advanced gastrointestinal cancer. The main side effect at a dose-limiting toxicity (DLT) of 350 mg/m² was neuropathy. Gastrointestinal and myelotoxicity were much lower than that observed with free l-OHP, yet a partial response was seen in 11% of the patients while 89% experienced a stable disease state for 4 months.⁴⁶⁾ Ye et al.⁴⁷⁾ prepared magnetic thermo-sensitive cationic liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) : DC-cholesterol : dimethyldioctadecyl ammonium bromide (DOAB) : cholesterol (17.08 : 0.81 : 0.9 : 1.21 as weight ratio) for the codelivery of l-OHP and antisense lucRNA of MDC1 (MDC1-AS). The codelivery of l-OHP and MDC1-AS by magnetic-thermosensitive cationic liposomes achieved the desirable thermosensitive release of l-OHP and MDC1-AS, that binds to UTR of MDC1 mRNA and increase its translation by inhibiting recognition of RNase. MDC1 regulates cell cycle checkpoints and causes cytotoxic effects on SiHa human uterus cells in vitro along with enhanced tumor-growth inhibitory effects in a SiHa tumor xenograft mouse model by i.v. injection, compared with the single drug delivery of either drug.⁴⁷⁾ Li et al.⁴⁸⁾ developed l-OHP-loading long-circulating thermosensitive liposomes (LCTLs). LCTLs are composed of DPPC : mPEG₂₀₀₀-DSPE (10 : 1 as weight ratio) and endowed with long-circulating property by PEGylation onto liposomes. When combined with heat, LCTLs enhanced the antitumor effects on 4T1 mouse mammary tumor-bearing models.⁴⁸⁾ Cheraga et al.⁴⁹⁾ prepared long-circulating PEGylated liposomes composed of EPC : cholesterol : mPEG₂₀₀₀-DSPE (2 : 1 : 0.2 M ratio) encapsulating l-OHP. They showed that the prepared l-OHP long-circulating liposomes improved the pharmacokinetic profiles of free l-OHP, but provided no data demonstrating the potency of such liposomes to produce antitumor effects.⁴⁹⁾

We have also developed a PEGylated liposomal l-OHP formulation composed of HSPC : cholesterol : mPEG₂₀₀₀-DSPE (2 : 1 : 0.1 M ratio) and assessed the therapeutic efficacy in a LLC tumor-bearing mouse model^50,51) and in a C26 murine colorectal carcinoma-bearing mouse model.⁵²⁾ Compared with free l-OHP, i.v. treatment with liposomal l-OHP clearly showed a higher level of therapeutic efficacy without any marked side effects.^50–52) We have also assessed the therapeutic efficacy of l-OHP liposome in three different murine tumor-bearing mouse models (LLC, C26, and B16BL6 mouse melanoma), in which a higher therapeutic effect of l-OHP liposome was observed in LLC tumor and C26 tumor than in B16BL6 tumor because of higher vascularization.⁵³⁾ We visualized the intratumor distribution of the encapsulated active pharmaceutical ingredient (API), i.e., l-OHP, via the use of microsynchrotron radiation X-ray fluorescence spectrometry (μ-SR-XRF), which enables identification and quantification of the chemical elements including the Pt in l-OHP.^54–56) In a DLD-1 human colorectal tumor xenograft mouse model, the Pt in PEGylated liposomal l-OHP was localized near the blood vessels, as manifested by the distribution of Fe in red blood cells, with higher intensity than that in free l-OHP.⁵⁷⁾ Repeated injections of PEGylated liposomal l-OHP markedly enhanced the tumor accumulation of a subsequent dose of l-OHP liposomes via improving the tumor vascular permeability,⁵⁷⁾ which is consistent with the results of our previous studies in that the distribution of liposomes was traced using fluorescent probe.^58,59)

We also evaluated the induction of peripheral neurotoxicity as manifested by the onset of hand-foot syndrome following the i.v. injection of liposomal l-OHP into rats and showed that liposomal l-OHP induces negligible skin toxicity, although l-OHP was somewhat accumulated in the hind paws.⁶⁰⁾ In addition, we investigated the phenomenon whereby an increased accumulation of l-OHP in lingual non-epithelial tissues by liposomal l-OHP exacerbates the free l-OHP-induced decrease in sweet taste sensitivity,⁶¹⁾ which suggests a regimen for cooling the tongue during treatment with liposomal l-OHP in clinical settings to prevent an exacerbation of the decrease in sweet taste sensitivity.

Furthermore, we found a unique property whereby PEGylated liposomal l-OHP modulates the tumor immune microenvironment⁶²⁾ and immune response to PEG.⁶³⁾ Immunomodulation of the tumor microenvironment is known to be an important factor that affects the therapeutic efficacy of many chemotherapeutic agents.⁶⁴⁾ Interestingly, the therapeutic effects of PEGylated liposomal l-OHP were substantially diminished in immunodeficient mice, compared with that in immunocompetent mice.⁶²⁾ In tumor xenograft immunocompetent mice, PEGylated liposomal l-OHP suppressed the incorporation of immune suppressor cells into tumor tissues. These include regulatory T cells (T_reg), myeloid-derived suppressor cells (MDSC), and tumor-associated macrophages (TAM), and this suppression preserved CD8⁺ T cell-mediated antitumor immunity.⁶²⁾ This indicates that PEGylated liposomal l-OHP exerts antitumor effects by orchestrating not only the inducement of a direct cytotoxic effect against tumor cells but also a modulation of the tumor immune microenvironment.

Our group and others have reported that PEGylated liposomes induce what is referred to as the “accelerated blood clearance (ABC) phenomenon” upon repeated injection.^65–67) Under such conditions, subsequent doses of PEGylated liposomes consequently lose their long-circulating characteristics. This phenomenon poses an impediment for the clinical application and use of PEGylated liposomal formulations. We confirmed that PEGylated liposomes including l-OHP attenuate the ABC phenomenon through suppression of the anti-PEG immunoglobulin M (IgM) response.⁶⁸⁾ In tumor-bearing mice, abrogation of the ABC phenomenon restores the intratumor accumulation of subsequently injected PEGylated liposomes. Accordingly, PEGylated liposomal l-OHP formulations afford another benefit in terms of abrogating the ABC phenomenon with the use of PEGylated liposomes.

In order to move liposomal l-OHP formulations into clinical development, stable formulation during storage, which replicates pharmacokinetics and therapeutic activity that is comparable to the original formulation, is indispensable. We assessed the stability of our liposomal l-OHP formulation during long-term storage (12 months at 2–8 °C). Consequently, the phospholipid composed of liposomes was degraded during storage, although the mean particle size was unchanged.⁶⁹⁾ To avoid such phospholipid degradation, we selected 2-morpholinoethansulfonic acid (MES) to adjust the pH of the l-OHP liposome solution to a neutral state. In the presence of MES, the liposomal l-OHP successfully maintained the characteristics needed to replicate in vivo pharmacokinetic profiles comparable to the original formulation after 12 months of storage at 2–8 °C.⁶⁹⁾

Taken together, PEGylated liposomal l-OHP formulation enhances the therapeutic efficacy of l-OHP, lessens the severe side effects of original l-OHP, and prevents occurrence of the ABC phenomenon with the use of PEGylated liposomes, which should help to overcome the problems associated with the clinical use of l-OHP and PEGylated liposomes.

3.3. Targeted Liposomal l-OHP Formulation

3.3.1. Transferrin-Modified Liposomes

Suzuki et al.⁷⁰⁾ developed a target-sensitive form of PEGylated liposome composed of DSPC : cholesterol : mPEG₂₀₀₀-DSPE (2 : 1 : 0.192 M ratio) wherein the transferrin (Tf) in l-OHP was coupled to surface-grafted PEG chains. Because Tf receptors (TfR) are overexpressed on the surface of many tumor cells, this pathway is expected to be effective for the targeted delivery of Tf-modified liposomes to tumor cells that overexpress TfR. The Tf-targeted liposomal l-OHP has achieved tumor-selective delivery of l-OHP in a C26-bearing mouse model and suppressed tumors at a rate that surpassed that of previous formulations such as l-OHP-containing PEGylated liposomes, l-OHP-containing classical (non-PEGylated) liposomes, and free l-OHP. The authors assumed that this potent antitumor efficacy of Tf-targeted liposomal l-OHP was due to the efficient delivery of l-OHP into the cytoplasm of tumor cells via TfR-mediated endocytosis after extravasation by the EPR effect. The advances of Tf-targeted liposomal l-OHP (MBP-426) were observed in a Phase II clinical trial for the treatment of gastric, gastroesophageal, and esophageal adenocarcinomas (NCT00964080).

3.3.2. Estrogen-Modified Liposomes

Sun et al.⁷¹⁾ prepared estrogen (ES)-modified PEGylated liposomes (ES-SSLs) that are composed of SPC : cholesterol : mPEG₂₀₀₀-DSPE : ES-PEG₂₀₀₀-DSPE (40 : 8 : 2.7 : 0.5 as weight ratio).⁷¹⁾ ES receptors are promising targets for various malignant tumors; thus, ES-SSLs can achieve selective accumulation into tumors via ES receptor-mediated targeted delivery. In in vitro study, Rhodamine B-loaded ES-SSLs were internalized into SGC-7901 human gastric cancer cells that highly express the ES receptors, and clearly were inhibited by co-incubation with ES recombinant proteins. In in vivo study, DiR-loaded ES-SSLs accumulated in the tumor of a SGC-7901 tumor xenograft mouse model, and l-OHP-loaded ES-SSLs significantly inhibited SGC-7901 tumor growth compared with either l-OHP-loaded SSLs or free l-OHP, and also lessened the severe toxicity caused by treatment with free-l-OHP.

3.3.3. PEGylated Cationic Liposomal l-OHP Formulation

We were challenged to develop a method that could deliver l-OHP to the blood vessels of tumors because targeted cytotoxic effects on tumor vessels could suppress their angiogenic processes and totally prevent the development and metastasis of primary tumor cells, which is a promising strategy for the destruction of solid tumors.^72–74) Tumor blood vessels are characterized by a unique physiological feature that involves negative charging, because anionic macromolecules such as glycoproteins, phospholipids, and proteoglycans are highly expressed on tumor blood vessels.^75–77) Therefore, we designed a “cationic” PEGylated liposomal l-OHP formulation composed of HSPC : cholesterol : DC-6-14 : mPEG₂₀₀₀-DSPE (2 : 1 : 0.2 : 0.2 M ratio) with the expectation that it could achieve an efficient targeting delivery of l-OHP to the tumor blood vessels, and we assessed the anti-angiogenic effects using a dorsal air sac mouse model.⁵¹⁾ The PEGylated cationic liposomal l-OHP exhibited a selective accumulation/binding to newly formed vessels and consequently suppressed the tumor angiogenesis.⁵¹⁾ We further investigated the therapeutic efficacy of the PEGylated cationic liposomal l-OHP in a Lewis lung carcinoma (LLC)-bearing mouse model.⁵⁰⁾ The PEGylated cationic liposomal l-OHP clearly suppressed tumor growth and increased the survival time of mice with no systemic side effects.⁵⁰⁾ In addition, in vitro studies indicated that PEGylated cationic liposomes were internalized not only by human umbilical vascular endothelial cells (HUVECs) but also by LLC cells.⁵⁰⁾ In an in vivo sequential dosing study, two sequential injections of PEGylated cationic liposomal l-OHP clearly enhanced the tumor accumulation of third dose of test liposomes, while a single injection of PEGylated cationic liposomal l-OHP did not enhance the accumulation of second dose of test liposomes.⁷⁸⁾ It appears that the two sequential injections of PEGylated cationic liposomal l-OHP induced higher apoptotic events in the tumor cells surrounding tumor blood vessels compared with a single injection and consequently expanded the intratumor distribution of the subsequent liposomes.⁷⁹⁾ These results suggest that our PEGylated cationic liposomal l-OHP achieves the dual targeting delivery of l-OHP to both tumor blood vessels and tumor cells, and consequently enhances therapeutic efficacy via the inducement of anti-angiogenic effects and cytotoxicity directly to tumor cells.

4. Conclusion

In our review, we concluded that l-OHP could be formulated in such a way as to have a better approach to decreasing the severe systemic side effects that are frequently expressed by treatment with the other Pt agents, such as cisplatin and carboplatin, although its use remains limited to clinical settings because of previously observed poor therapeutic outcomes and other cumulative side effects. In order to overcome such issues, several DDS techniques have been reported in both basic and clinical studies. We developed a PEGylated liposomal l-OHP formulation that demonstrates good antitumor potency on tumor-bearing animal models with no severe side effects from selective delivery to tumors via the EPR effect. Compared to the other DDS techniques for l-OHP, our l-OHP liposome could be a feasible formulation to preclinical/clinical studies because its high stability during long-term storage has been already confirmed and the Good Manufacturing Practice (GMP)-grade large-scale manufacturing system with high recovery ratio has already been established. Accordingly, our l-OHP formulation could be applicable to clinical use and could become a viable alternative to free l-OHP for the treatment of cancer patients, which would improve their QOL.

Acknowledgments

This work was partially supported by the Tokushima University UZUSHIO Project and a research program for the development of an intelligent Tokushima artificial exosome (iTEX) from Tokushima University.

Conflict of Interest

The authors declare no conflict of interest.

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